Engineering kinetics of TLR7/8 agonist release from bottlebrush prodrugs enables tumor-focused immune stimulation

Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 “bottlebrush prodrugs” (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.


The PDF file includes:
Supplementary Materials and Methods Figs. S1 to 18 Tables S1 to S3 Legend for table S4 Other Supplementary Material for this manuscript includes the following:

Materials
Solvents used are of HPLC grade, purchased from Millipore Sigma, and used as received unless otherwise noted. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc.

Mass spectrometry (MS)
Low resolution liquid chromatography mass spectrometry (LC-MS) was performed using an Agilent 6125B mass spectrometer attached to an Agilent 1260 Infinity LC. It utilizes an electrospray (ESI) source.
ChemStation acquisition and data analysis software is used. High resolution mass spectrometry (HR-MS) measurements were recorded using a JEOL AccuTOF 4G LC-plus system equipped with an ionSense DART (Direct Analysis in Real Time) source. The system operates with an accuracy of 5 ppm and a resolving power >10,000 (FWHM). msAxel was used as the acquisition and data processing software.

Size Exclusion Chromatography (SEC)
SEC characterization was done using an Agilent 1260 LC system equipped with a Wyatt T-rEX refractive index detector and Wyatt DAWN HELEOS 18 angle light scattering detector. R848-MMs and R848-BPDs were run on Agilent PL1110-6500 columns in tandem at a temperature of 60 o C and flow rate of 1 mL/min with dimethyl formamide (DMF) containing 0.025 M LiBr as the eluent.

Dynamic Light Scattering (DLS)
DLS was performed using a Wyatt Dyna Pro Plate Reader. BPD suspensions were prepared in a solution of nanopure water (MilliQ) (1mg/ml). Measurements were made in sets of 10 acquisitions. The average hydrodynamic diameters were calculated by using the DLS correlation function via a regularization fitting method (Dynamics 7.4.0.72 software package from Wyatt Technology).

Preparative gel-permeation chromatography (prep-GPC)
prep-GPC was performed on a JAI Preparative Recycling HPLC (LaboACE-LC-5060) system equipped with 2.5HR and 2HR columns in series (20 mm ID x 600 mm length) using CHCl3 as the eluent.

Small Molecule Syntheses
Esterification General procedure. Compounds prepared using an adapted procedure from Vohidov et al. 32 To an oven-dried Schlenk flask was added 6-bromohexanoic acid (1.05 equiv) or 3-(2-(2-(2bromoethoxy)ethoxy)ethoxy)propanoic acid (bromo-PEG3-acid, 1.05 equiv), EDC•HCl (1.10 equiv), and DMAP (0.10 equiv), and the solids placed under an N2 atmosphere. Dry DCM (10 mL) was then added, and reaction left to stir at room temperature for 5 min. The appropriate benzaldehyde (1.0 equiv) was added against the flow of N2, following which the reaction mixture was stirred at room temperature for 18 h. H2O (10 mL) was added to quench the reaction, and the aqueous layer was washed with DCM (3 x 10 mL). The combined organics were dried using Mg2SO4, filtered, and the solvent removed under reduced pressure. The crude product was purified using column chromatograph (silica, 9:1 DCM:EtOAc).  170.9, 152.1, 145.1, 135.3, 124.8, 123.5, 110.9, 56.2, 33.8, 33.6, 32.4, 27 atmosphere. Dry THF (10 mL), and dry MeOH (10 mL) were added and the reaction vessel was immersed in an ice bath. Against the flow of N2, NaBH4 (1.5 equiv) was added in three portions as a solid, and the reaction left to stir at 0 °C for 15 min. Following this, H2O (50 mL) was added, and the reaction mixture was extracted with DCM (3 x 20 mL). The combined organics were washed with brine (1 x 50 mL), dried with Mg2SO4, filtered, and the solvent removed in vacuo. The crude compound was purified using column chromatography (silica, 100% DCM to 1:1 DCM:EtOAc gradient). General procedure. Compounds prepared using an adapted procedure from Vohidov et al. 1 To an ovendried Schlenk flask was added NaN3 (1.5 equiv), NaI (0.1 equiv), and the flask placed under an N2 atmosphere. Dry DMF (5 mL) was added, and the reaction stirred at room temperature for 5 min. In a separate flask, the appropriate compound 2a-f was dissolved in dry DMF (10 mL), and this solution was added to the reaction mixture via cannula. The reaction was left to stir at room temperature for 16 h.
Following this, EtOAc (40 mL) was added, yielding a white precipitate, and the organic layer was washed with water (3 x 500 mL) and brine (1 x 500 mL). The organic layer was dried over Mg2SO4, filtered, and the solvent removed under reduced pressure. The crude compound was then purified using column chromatography (silica, 100% hexanes to 6:4 EtOAc:hexanes gradient).